(1) Soluble (100,000 x g supernatant) guanylate cyclase from guinea pig kidney and liver was activated 3-4 fold by 0.1-0.2 mM Co ions. None of a number of other metal ions substituted for Co ions. Basal activity increased linearly as assay pH increased from 5.9-9; in the presence of Co ions activity was optimal from pH 7.3-7.8. Incubation of fresh supernatants at 37 degrees or dialysis at 0 degrees C increased cyclase activity and decreased the magnitude of cobalt activation. After elution from DEAE Bio-Gel columns, the enzyme was not activated by Co ions. (2) Kidney and liver supernatants contain a heat stable dialyzable inhibitor of guanylate cyclase. This material elutes from Sephadex G-25 columns with a Kd of 0.6; it does not affect the fresh soluble cyclase but does inhibit the enzyme after dialysis or elution from a Bio-Gel P-100 column. Inhibition is proportional to the amount of G-25 material added and is completely reversed by Co ions. Co ions activation of fresh supernatant enzyme may be related to Co ions counteracting the effect of the heat stable inhibitor. (3) Guanylate cyclase has been purified approx. 500-fold from rat liver supernatant using ammonium sulfate fractionation, anion exchange, gel filtration and affinity chromatography, sucrose density gradient centrifugation and preparative disc gel electrophoresis. BIBLIOGRAPHIC REFERENCES: Clyman, R.I., Manganiello, V.C., and Vaughan, M.: Oxygen: Influence on cyclic nucleotides and calcium sequestration in the human umbilical artery. Chest 71S: 247-249, 1977. Ting, C.C., Tsai, S.-C., and Rogers, M.J.: Host control of tumor growth. Science 197: 571-3, 1977.